The Vascular Biochemistry and Molecular Biology Core is designed to provide investigators with access to routine as well as state-of-the-art molecular and biochemical tools, expertise, and techniques. The central theme of this Program Project is that exercise induces beneficial phenotypic changes in vascular cells. Our long-term objectives are to advance the understanding of the molecular, biochemical, cellular, and integrative mechanisms that underlie these training effects. The application of molecular and biochemical techniques will be integral in the exploration of the mechanisms underlying the physiological observations made by this PPG group. The Core's activity reflects a centralization and expansion of services that the Core Leader has successfully provided to PPG investigators for the past decade. The Core laboratory is fully equipped and dedicated to address the stated goals and is directed and staffed by well-qualified personnel. The following Aims are based on the needs of the PPG investigators and reflect a compilation of procedures and techniques that will ensure the efficient completion of each PPG project. The long-term goal of this Core is to track the expression and modulation of specific proteins throughout the coronary and peripheral vasculature and to demonstrate an effect of exercise and/or high-fat-cholesterol on these features. Aim 1 is to perform reverse transcriptase-PCR (RT-PCR) to detect specific mRNAs expressed throughout the vasculature. Aim 2 is to perform real-time PCR in order to more accurately quantitate mRNA expression in select samples from the vasculature of control and experimental animals. Aim 3 is to utilize immunoblots and co-immunoprecipitations to detect and relatively quantitate specific proteins and interactions expressed throughout the circulation. Aim 4a is to use in situ PCR to demonstrate tissue-specific expression of select mRNA species. Aim 4b is to use laser-capture microdissection to selectively isolate and recover select cells from sections of vascular tissue. Real-time PCR will then be used to assess mRNA levels in the recovered cells. Aim 5 is to generate two commercial libraries (one from conduit arteries and one from resistance arterioles) and screen for cDNAs encoding select proteins from the coronary vasculature. Aim 6 is to prepare adeno-associated viral constructs for the transduction of various proteins and siRNA into vascular cells. Aim 7 is to coordinate the commercial preparation of select porcine-specific antibodies. Aim 8 is to prepare and analyze peripheral blood cells from transgenic and non-transgenic pigs. Aim 9 is to provide additional standard techniques as well as pioneer new methods as a resource for PPG investigators.